本文關(guān)鍵詞： 胰島素受體底物2 周期蛋白依賴激酶抑制因子1A 多囊卵巢綜合征 微小RNA 單核苷酸多態(tài)性　出處：《暨南大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：多囊卵巢綜合征(PCOS)是一種常見的以月經(jīng)紊亂、持續(xù)性排卵障礙和卵巢多囊樣改變?yōu)橹饕R床特征,常合并高雄激素血癥(HA)和胰島素抵抗(IR)及代償性的高胰島素血癥(HI)的生殖功能障礙與代謝異常并存的內(nèi)分泌代謝紊亂綜合癥。PCOS屬于生殖系統(tǒng)和內(nèi)分泌代謝性疾病,越來越多的研究表明PCOS是一種累及全身、危害女性終生健康的基因與環(huán)境共同作用的遺傳相關(guān)性疾病。目前有關(guān)PCOS病因和發(fā)病機理的研究有諸多報道,但其病因和確切發(fā)病機制仍不十分清楚。PCOS發(fā)病常常表現(xiàn)為家族聚集性,諸多學(xué)者認(rèn)為遺傳因素在PCOS病因?qū)W方面起重要作用;但近年P(guān)COS相關(guān)遺傳候選基因研究仍無突破性進(jìn)展。目前普遍認(rèn)為PCOS是遺傳基因與環(huán)境因素相互作用的結(jié)果,近年的研究關(guān)注點在表觀遺傳學(xué)方面領(lǐng)域,研究的方法集中在DNA甲基化方面。隨著分子生物技術(shù)的發(fā)展,尤其是生物信息學(xué)的廣泛應(yīng)用,研究的關(guān)注點開始轉(zhuǎn)向非編碼微小RNA(miRNA)與PCOS發(fā)生發(fā)展中的關(guān)系,已有研究表明miRNA在PCOS發(fā)生過程中參與調(diào)控作用。miRNA是一種微小片段非編碼RNA,主要通過與靶信使RNA(mRNA)的3’UTR區(qū)域特異性結(jié)合,在靶目標(biāo)mRNA的降解、翻譯抑制和脫腺苷化等轉(zhuǎn)錄后水平調(diào)控細(xì)胞基因的轉(zhuǎn)錄及表達(dá),進(jìn)而參與了生命或疾病的發(fā)生過程。盡管其不參與基因的編碼,但是通過與靶RNA結(jié)合來降低其穩(wěn)定性或下調(diào)它們的轉(zhuǎn)錄,進(jìn)而參與到細(xì)胞生長、分化發(fā)育、凋亡等生物學(xué)過程中。而miRNA與其靶基因結(jié)合區(qū)域單核苷酸多態(tài)性(SNPs)與多種疾病易感性密切相關(guān)。胰島素抵抗是PCOS的基本病理生理特征之一,研究認(rèn)為IRS-2基因多態(tài)在胰島素抵抗中扮演了重要作用,PCOS患者卵泡液中miR-135a表達(dá)上調(diào),而潛在的miRNA目標(biāo)基因IRS-2的表達(dá)下調(diào)。PCOS發(fā)生的另一個重要機制是卵巢局部微環(huán)境的改變,研究發(fā)現(xiàn),miR-93以及其靶基因CDKN1A共同調(diào)節(jié)PCOS卵巢顆粒細(xì)胞的生理功能通過調(diào)節(jié)卵巢局部細(xì)胞因子,參與多囊卵巢的形成。目前,關(guān)于miRNA下游靶基因胰島素受體底物2基因(IRS2)和周期蛋白依賴激酶抑制因子1A基因(CDKN1A/P21)結(jié)合區(qū)域SNPs與PCOS病變在人群中的關(guān)聯(lián)研究報道暫時未見�；趥鹘y(tǒng)的分子遺傳學(xué)原理,結(jié)合表觀遺傳學(xué)的研究手段,本研究根據(jù)現(xiàn)有的相關(guān)基因的研究成果,從生物醫(yī)學(xué)文獻(xiàn)數(shù)據(jù)庫篩選,采用生物信息學(xué)方法分析和預(yù)測PCOS相關(guān)的mi RNA靶結(jié)合區(qū)域內(nèi)的單核苷酸多態(tài)性位點,運用先進(jìn)的高通量基因測序技術(shù)進(jìn)行測序,比較分析所研究對象的單核苷酸多態(tài)性位點、基因型等差異,探討IRS2、CDKN1A基因3’UTR區(qū)域特異性結(jié)合單核苷酸多態(tài)性與PCOS相關(guān)病變的高度異質(zhì)性、家族聚集現(xiàn)象之間的相關(guān)性,為進(jìn)一步闡明pcos的發(fā)病機制、預(yù)測其發(fā)生風(fēng)險,以及為pcos的臨床早期防治、診斷和病情評估及預(yù)后判斷提供新的參考依據(jù)。本研究采用病例-對照研究方法,通過臨床上收集pcos患者及對照組的臨床信息資料及外周血樣本,描述臨床上的基本特征及生活習(xí)慣,分析臨床特征與基因多態(tài)性、基因型的關(guān)系。研究目的1.通過收集pcos患者和對照組臨床基本信息以及輔助檢查的數(shù)據(jù)資料,描述和分析pcos在人群當(dāng)中的臨床基本特征。2.通過篩選irs2、cdkn1a基因的mirnas靶結(jié)合區(qū)域內(nèi)的單核苷酸多態(tài)性位點,探討遺傳多態(tài)性位點、基因型與pcos病變的相關(guān)性,從分子水平發(fā)現(xiàn)pcos的易感因素及潛在的危險因素。3.利用生物信息學(xué)方法,從表觀遺傳學(xué)角度分析基因-基因以及基因-環(huán)境之間的聯(lián)合作用,闡明基因-基因以及基因-環(huán)境之間的相互作用,探討單核苷酸多態(tài)性與pcos臨床特征、異質(zhì)性和家族聚集現(xiàn)象的關(guān)系。研究方法1.采用病例對照研究設(shè)計,選取2014年08月—2015年10月在廣東省計劃生育�？漆t(yī)院門診就診及住院的共188例pcos患者及對照組為研究對象,部分收集5mledta抗凝血,抽提全基因組dna,進(jìn)行snps基因型檢測。2.運用問卷調(diào)查方法收集調(diào)查對象基本信息及相關(guān)暴露因素。填寫調(diào)查問卷,對其一般特征及臨床資料進(jìn)行回顧性分層對照分析,采用elisa法檢測生殖相關(guān)激素,并計算卵泡刺激素/黃體生成素(fsh/lh)比值;測定空腹胰島素(fins)及空腹血糖(fpg)值,并計算homa-ir指數(shù);測定抗苗勒管激素;并予以陰道b超等輔助檢查。3.運用spss13.0軟件比較分析mir-135a、mir-93靶基因irs-2、cdkn1a結(jié)合區(qū)單核苷酸多態(tài)性與pcos及其臨床特征相關(guān)性。研究結(jié)果1.臨床資料回顧性分析1)pcos患者的bmi、基礎(chǔ)lh、基礎(chǔ)t、amh、homa-ir顯著高與對照組(p0.05)。2)高雄激素血癥組fins水平顯著高于非高雄激素血癥組(p0.05);hi組bmi、prl、fins顯著高于非hi組(p0.05);肥胖組homa-ir水平顯著高于非肥胖組,差異有統(tǒng)計學(xué)意義(p0.05);月經(jīng)稀發(fā)組amh、homa-ir顯著高于月經(jīng)正常組,差異有統(tǒng)計學(xué)意義(p0.05)。2.共篩選了miRNA相關(guān)的2個靶基因7個單核苷酸多態(tài)性位點,其中IRS2基因兩個位點:rs2289046和rs1865434;CDKN1A基因5個位點:rs1059234、rs3176366、rs3176337、rs3176359和rs74801436。對照組中的基因型rs2289046、rs1865434、rs1059234、rs3176337分布符合哈溫平衡。另外3個單核苷酸多態(tài)性位點在本樣本群體中不是多態(tài)的。3.IRS2基因2個單核苷酸多態(tài)性位點與CDKN1A基因2個單核苷酸多態(tài)性位點的三種基因型和兩種等位基因頻率分布在PCOS組和對照組以及PCOS組內(nèi)的分層比較中的差異無統(tǒng)計學(xué)意義(P0.05)。研究結(jié)論1.PCOS患者臨床特征多樣化,符合PCOS臨床診斷標(biāo)準(zhǔn),表現(xiàn)異質(zhì)性;胰島素抵抗的發(fā)生與月經(jīng)稀發(fā)和肥胖有關(guān)。2.暫未發(fā)現(xiàn)篩選的IRS2基因、CDKN1A基因miRNA靶結(jié)合區(qū)域的單核苷酸多態(tài)性位點與PCOS病變的遺傳易感性具有顯著相關(guān)性;調(diào)整年齡、BMI、FINS等因素后,兩組之間仍未見顯著相關(guān)性。
[Abstract]:Polycystic ovary syndrome (PCOS) is a kind of common to menstrual disorders, persistent anovulation and polycystic ovaries as the main clinical features, often associated with Kaohsiung hormones (HA) and insulin resistance (IR) and compensatory hyperinsulinemia (HI).PCOS metabolic disorder syndrome coexisting abnormal reproductive function the metabolic disorder and belongs to the reproductive system and the endocrine and metabolic disease, more and more studies show that PCOS is a systemic disease, genetic correlation between the harm to women's lifelong health gene environment interaction. The present research on PCOS etiology and pathogenesis of the disease there are many reports, but its etiology and pathogenesis is still not very clear the incidence of.PCOS is often a family gathering, many scholars believe that genetic factors play an important role in the etiology of PCOS; but in recent years, genetic research on genes related to PCOS is still no candidate Breakthrough. PCOS is considered to be the interaction of genetic and environmental factors, in recent years the research focus in the field of epigenetics, research methods focus on DNA methylation. With the development of molecular biological technology, especially the widespread application of bioinformatics to non encoding micro RNA began to study focus (miRNA) and PCOS in the development of the relationship, studies have shown that miRNA in the process of PCOS in.MiRNA regulation is a tiny fragment encoding RNA, mainly through the target messenger RNA (mRNA) 3 'UTR region specific binding in the degradation of target mRNA, transcription and expression translation inhibition and deadenylation and post transcriptional regulation of cell gene, and then participate in the process of life or disease. Although it is not involved in the gene encoding, but by binding to RNA to reduce its stability The qualitative or downregulation of their transcription, and then participate in cell growth, differentiation, apoptosis and other biological processes. MiRNA and its target gene with single nucleotide polymorphisms (SNPs) associated with various disease susceptibility. Insulin resistance is the basic pathophysiological features of PCOS, studies suggest that IRS-2 gene plays an important role in insulin resistance in patients with PCOS, upregulate the expression of miR-135a in follicular fluid and the expression of miRNA target gene IRS-2 potential down another important mechanism of.PCOS is the ovarian microenvironment, the study found that the physiological function of miR-93 and its target gene CDKN1A regulated PCOS ovarian granulosa cells by regulating ovarian cytokine, involved in the formation of polycystic ovary. At present, about miRNA downstream target genes of insulin receptor substrate 2 gene (IRS2) and cyclin dependent kinase inhibitor Gene 1A (CDKN1A/P21) SNPs and PCOS combined with the regional association of disease in the population reported no temporary. Based on the traditional principle of molecular genetics, combined with research methods of genetics, based on the research results of the existing related genes, screened from the biomedical literature database, using bioinformatics method to analyze and forecast PCOS the MI RNA target binding sites of single nucleotide polymorphisms within the region, were sequenced using high-throughput gene sequencing technology, comparative analysis of single nucleotide polymorphism of the object of study, genotype differences of IRS2, CDKN1A gene 3 'UTR region specific with high heterogeneity and single nucleotide polymorphism of PCOS related diseases. The correlation between the phenomenon of familial aggregation, to further elucidate the pathogenesis of PCOS, predict the risk of early prevention and treatment of PCOS and clinical diagnosis. Provide new reference for judging the evaluation and prognosis of fault and disease. In this study, a case-control study, PCOS patients and control subjects were collected by clinical data and clinical information of peripheral blood samples, describes the basic characteristics and habits and clinical analysis of clinical features and gene polymorphism, genotype. Objective: 1. patients with PCOS and control group by collecting clinical data of basic information and auxiliary examination, description and analysis of the clinical characteristics of.2. PCOS in the crowd through the screening of IRS2, miRNAs target gene CDKN1A binding sites of single nucleotide polymorphisms within the region, to explore the genetic polymorphism, relationship between genotype and PCOS lesions. From the molecular level to find the susceptible factors of PCOS and the potential risk factors of.3. using bioinformatics methods, from the perspective of epigenetics analysis of gene gene and gene environment Combined effects, elucidate the interactions between gene gene and gene environment, to investigate single nucleotide polymorphism and PCOS clinical features, the relationship between the phenomenon of heterogeneity and familial aggregation. Methods 1. case-control study from 2014 08 months to October 2015 in Guangzhou Family Planning Specialty Hospital outpatient clinic and hospitalized a total of 188 patients with PCOS and the control group as the research object, part of the collection of 5mledta anticoagulant, extraction of genomic DNA and SNPs genotypes of.2. using the questionnaire survey method to collect the basic information survey and related exposure factors. Fill in the questionnaire, the general characteristics and clinical data were retrospectively stratified control analysis, ELISA method was used to detect reproductive hormones, and the calculation of FSH / LH ratio (fsh/lh); fasting insulin (fins) and fasting blood glucose (FPG), and calculate the HOMA-IR index; Determination of anti Mullerian hormone; and vaginal examination using.3. B Ultrasound SPSS13.0 software analysis and comparison of mir-135a, mir-93 and IRS-2 target gene, CDKN1A binding between single nucleotide polymorphism of PCOS and its clinical features. Results of the 1. clinical data were retrospectively analyzed in 1 patients with PCOS) BMI, LH, t AMH, HOMA-IR, and the control group was significantly higher (P0.05).2) Kaohsiung hormones group fins level was significantly higher than that of non Kaohsiung hormones group (P0.05); group hi BMI, PRL, fins were significantly higher than that of HI group (P0.05); the level of HOMA-IR in obese group was significantly higher than that in the non obese group, the difference was statistically significant (P0.05) oligomenorrhea; group AMH, HOMA-IR group was significantly higher than that of normal menstruation, the difference was statistically significant (P0.05.2.) were screened miRNA 2 related target gene 7 SNPs, including two SNPs of IRS2 gene: rs2289046 and rs1865434; CDKN1A gene 5 sites: rs1059234, rs3176366, rs3176337, rs3176359 and rs74801436. genotype rs2289046, control group rs1865434, rs1059234, rs3176337 distribution is in accordance with Hardy Weinberg equilibrium. Three genotypes of 2 SNPs of CDKN1A gene and 3 other SNPs are not polymorphic in this sample of.3.IRS2 gene 2 nucleotide polymorphic loci and two had no statistical significance in the PCOS group and the control group and the difference between the layers in PCOS group in the gene frequency distribution (P0.05). Conclusions: 1.PCOS patients clinical characteristics of diversification, conforms to the standard of clinical diagnosis of PCOS, showing the heterogeneity; the occurrence of insulin resistance and oligomenorrhea and obesity about.2. not found IRS2 gene screening, CDKN1A gene with genetic susceptibility to miRNA target region single nucleotide polymorphism and PCOS disease have significant correlation There was no significant correlation between the two groups after the adjustment of age, BMI, FINS and other factors.

【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R711.75

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